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Pathological vascular remodeling is the underlying cause of atherosclerosis and abdominal aortic aneurysm (AAA). Here, we analyzed the role of galectin-1 (Gal-1), a β-galactoside-binding protein, as a therapeutic target for atherosclerosis and AAA. Mice lacking Gal-1 (Lgals1-/-) developed severe atherosclerosis induced by pAAV/D377Y-mPCSK9 adenovirus and displayed higher lipid levels and lower expression of contractile markers of vascular smooth muscle cells (VSMCs) in plaques than wild-type mice. Proteomic analysis of Lgals1-/- aortas showed changes in markers of VSMC phenotypic switch and altered composition of mitochondrial proteins. Mechanistically, Gal-1 silencing resulted in increased foam cell formation and mitochondrial dysfunction in VSMCs, while treatment with recombinant Gal-1 (rGal-1) prevented these effects. Furthermore, rGal-1 treatment attenuated atherosclerosis and elastase-induced AAA, leading to higher contractile VSMCs in aortic tissues. Gal-1 expression decreased in human atheroma and AAA compared to control tissue. Thus, Gal-1-driven circuits emerge as potential therapeutic strategies in atherosclerosis and AAA. This study was funded by the Spanish MINECO (PID2019-106814RB-I00 and PGC2018-097019-B-I00), CAM (Complemento II-CM, S2017/BMD-3673), Fondo de Investigaciones Sanitarias ISCiii-FEDER [Biobancos RD09/0076/00101 and PRB3 (IPT17/0019, ProteoRed)], and FEDER “Una manera de hacer Europa.” CIBERCV and CIBERDEM are Instituto de Salud Carlos III projects. This work also received funding from “La Caixa” Banking Foundation under the project code HR17-00247. The CNIC is supported by Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia, Innovación y Universidades (MCNU), and Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Further support was provided by Santander-UAM, Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación (PICT 2010-870, Argentina), Fundación Sales, and Fundación Bunge y Born (Argentina). We thank the support of Ferioli, Ostry, and Caraballo families. Sí

Characterizing the diverse melon cultivars for nutrition aids in crop improvement and promoting a healthy diet. Here, we used in vitro assays to characterize the nutritional qualities and health-beneficial effects of 30 melon (Cucumis melo L.) genotypes, including 10 improved cultivars, 16 landraces, and 4 wild types collected from different parts of India. Two landraces (Sidoota and Alper Green) had the highest (12.20 and 11.25) total soluble solids (TSS) contents. The Sidoota and Pappusa landraces had high reducing sugar contents (2.84 and 2.81 mg g−1 fresh weight [FW]). The highest polyphenols contents (22.0 mg g−1 FW) were observed in the landraces Mage Kaayi-2, Budamekaayi, and Small Melon. Reflecting on the primary and secondary metabolite contents, the Mekke Kaayi and Giriyala landraces exhibited high 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (97.6 and 91% at 100 μg mL−1). Additionally, seven of the landraces showed significant nitric oxide (NO) induction activity (&gt, 80% inhibition at 200 μg mL−1), indicating their potential health benefits, and seven showed considerable angiotensin-converting enzyme (ACE) inhibition activity (highest in Kashi Madhu), indicating their potential usefulness in reducing hypertension. Genotypes with high health beneficial compounds identified in this study can be used for breeding improved melon cultivars to promote these fruits as well as a healthy diet.

Fusarins are a class of mycotoxins of the polyketide family produced by different Fusarium species, including the gibberellin-producing fungus Fusarium fujikuroi . Based on sequence comparisons between polyketide synthase (PKS) enzymes for fusarin production in other Fusarium strains, we have identified the F. fujikuroi orthologue, called fusA . The participation of fusA in fusarin biosynthesis was demonstrated by targeted mutagenesis. Fusarin production is transiently stimulated by nitrogen availability in this fungus, a regulation paralleled by the fusA mRNA levels in the cell. Illumination of the cultures results in a reduction of the fusarin content, an effect partially explained by a high sensitivity of these compounds to light. Mutants of the fusA gene exhibit no external phenotypic alterations, including morphology and conidiation, except for a lack of the characteristic yellow and/or orange pigmentation of fusarins. Moreover, the fusA mutants are less efficient than the wild type at degrading cellophane on agar cultures, a trait associated with pathogenesis functions in Fusarium oxysporum . The fusA mutants, however, are not affected in their capacities to grow on plant tissues.

Tombusviruses express a 19 kDa protein (p19) that, as a dimeric protein, suppresses the RNAs silencing pathway during infection by binding short-interfering RNA (siRNA) and preventing their association with the RNA-induced silencing complex (RISC). The p19 protein can bind to both endogenous and synthetic siRNAs with a high degree of size selectivity but with little sequence dependence. It also binds to other endogenous small RNAs such as microRNAs (miRNAs) but with lower affinity than to canonical siRNAs. It has become apparent, however, that miRNAs play a large role in gene regulation; their influence extends to expression and processing that affects virtually all eukaryotic processes. In order to develop new tools to study endogenous small RNAs, proteins that suppress specific miRNAs are required. Herein we describe mutational analysis of the p19 binding surface with the aim of creating p19 mutants with increased affinity for miR-122. By site-directed mutagenesis of a single residue, we describe p19 mutants with a nearly 50-fold increased affinity for miR-122 without altering the affinity for siRNA. Upon further mutational analysis of this site, we postulate that the higher affinity relies on hydrogen-bonding interactions but can be sterically hindered by residues with bulky side chains. Finally, we demonstrate the effectiveness of a mutant p19, p19-T111S, at sequestering miR-122 in human hepatoma cell lines, as compared to wild-type p19. Overall, our results suggest that p19 can be engineered to enhance its affinity toward specific small RNA molecules, particularly noncanonical miRNAs that are distinguishable based on locations of base-pair mismatches. The p19-T111S mutant also represents a new tool for the study of the function of miR-122 in post-transcriptional silencing in the human liver. © 2011 American Chemical Society.

Xavi Carpena et al., In heme enzymes belonging to the peroxidase-cyclooxygenase superfamily the proximal histidine is in close interaction with a fully conserved asparagine. The crystal structure of a mixture of glycoforms of myeloperoxidase (MPO) purified from granules of human leukocytes prompted us to revise the orientation of this asparagine and the protonation status of the proximal histidine. The data we present contrast with previous MPO structures, but are strongly supported by molecular dynamics simulations. Moreover, comprehensive analysis of published lactoperoxidase structures suggest that the described proximal heme architecture is a general structural feature of animal heme peroxidases. Its importance is underlined by the fact that the MPO variant N421D, recombinantly expressed in mammalian cell lines, exhibited modified spectral properties and diminished catalytic activity compared with wild-type recombinant MPO. It completely lost its ability to oxidize chloride to hypochlorous acid, which is a characteristic feature of MPO and essential for its role in host defense. The presented crystal structure of MPO revealed further important differences compared with the published structures including the extent of glycosylation, interaction between light and heavy polypeptides, as well as heme to protein covalent bonds. These data are discussed with respect to biosynthesis and post-translational maturation of MPO as well as to its peculiar biochemical and biophysical properties. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported by Spanish Ministerio de Educación y Ciencia Grants FIS2008-03845 and BIO2005-08686-C02-01, Generalitat de Catalunya Grants 2005SGR-00112 and 2005SGR-00036, and Austrian Science Fund FWF project P20664 (to P. G. F.). This work was also supported by a grant from the I3P program of the Spanish Research Council (to X. C.)

Addition of affinity tags to bacteriophage particles facilitates a variety of applications, including vaccine construction and diagnosis of bacterial infections. Addition of tags to phage capsids is desirable, as modification of the tails can lead to poor adsorption and loss of infectivity. Although tags can readily be included as fusions to head decoration proteins, many phages do not have decoration proteins as virion components. The addition of a small (10-amino-acid) Strep-tag II (STAG II) to the mycobacteriophage TM4 capsid subunit, gp9, was not tolerated as a genetically homogenous recombinant phage but could be incorporated into the head by growth of wild-type phage on a host expressing the capsid-STAG fusion. Particles with capsids composed of wild-type and STAG-tagged subunit mixtures could be grown to high titers, showed good infectivities, and could be used to isolate phage-bacterium complexes. Preparation of a STAG-labeled fluoromycobacteriophage enabled capture of bacterial complexes and identification of infected bacteria by fluorescence. Fil: Piuri, Mariana. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Rondon Salazar, Liliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Urdániz, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Hatfull, Graham F.. University of Pittsburgh; Estados Unidos

A recent experiment has probed the electron transfer kinetics in the early stage of photosynthesis in Rhodobacter sphaeroides for the reaction center of wild type and different mutants [Science 316, 747 (2007)]. By monitoring the changes in the transient absorption of the donor-acceptor pair at 280 and 930 nm, both of which show non-exponential temporal decay, the experiment has provided a strong evidence that the initial electron transfer kinetics is modulated by the dynamics of protein backbone. In this work, we present a model where the electron transfer kinetics of the donor-acceptor pair is described along the reaction coordinate associated with the distance fluctuations in a protein backbone. The stochastic evolution of the reaction coordinate is described in terms of a non-Markovian generalized Langevin equation with a memory kernel and Gaussian colored noise, both of which are completely described in terms of the microscopics of the protein normal modes. This model provides excellent fits to the transient absorption signals at 280 and 930 nm associated with protein distance fluctuations and protein dynamics modulated electron transfer reaction, respectively. In contrast to previous models, the present work explains the microscopic origins of the non-exponential decay of the transient absorption curve at 280 nm in terms of multiple time scales of relaxation of the protein normal modes. Dynamic disorder in the reaction pathway due to protein conformational fluctuations which occur on time scales slower than or comparable to the electron transfer kinetics explains the microscopic origin of the non-exponential nature of the transient absorption decay at 930 nm. The theoretical estimates for the relative driving force for five different mutants are in close agreement with the experimental estimates obtained using electrochemical measurements. � 2013 American Institute of Physics.

The β-1,4-endoglucanase (EC 3.2.1.4) from the hyperthermophilic archaeon Pyrococcus horikoshii (EGPh) has strong hydrolyzing activity toward crystalline cellulose. When EGPh is used in combination with β-glucosidase (EC 3.2.1.21), cellulose is completely hydrolyzed to glucose at high temperature, suggesting great potential for EGPh in bioethanol industrial applications. The crystal structure of EGPh shows a triosephosphate isomerase (TIM) (β/α) 8 -barrel fold with an N-terminal antiparallel β-sheet at the opposite side of the active site and a very short C-terminal sequence outside of the barrel structure. We describe here the function of the peripheral sequences outside of the TIM barrel core structure. Sequential deletions were performed from both N and C termini. The activity, thermostability, and pH stability of the expressed mutants were assessed and compared to the wild-type EGPh enzyme. Our results demonstrate that the TIM barrel core is essential for enzyme activity and that the N-terminal β-sheet is critical for enzyme thermostability. Bioinformatics analyses identified potential key residues which may contribute to enzyme hyperthermostability.

The bacterial toxin pyolysin (PLO) belongs to the family of cholesterol-dependent cytolysins (CDCs), which form large, ring-shaped oligomeric pores in cholesterol-containing membranes. Monomeric CDC molecules have a structure of four domains, with domains 2 and 3 packed against each other. After binding to target membranes containing cholesterol, toxin monomers oligomerize into pre-pore complexes. Trans-membrane pores form when the pre-pores insert into the lipid bilayer. Membrane insertion requires each subunit in the pre-pore to undergo a significant change in conformation, including the separation of domains 2 and 3. We here characterize a pyolysin mutant with an engineered disulfide bond between domains 2 and 3. The disulfide-tethered mutant binds to membranes but does not form oligomers. When mixed with wild type PLO, the two proteins form hybrid oligomers, which are reduced in size and arc-shaped rather than ring-shaped. With equimolar mixtures or the disulfide mutant in slight excess, the hybrid oligomers retain pore-forming activity, while a larger excess of the mutant suppresses pore formation. These results support a "partially cooperative" mode of protein activity, in which a limited number of functional subunits within an oligomer have to cooperate to initiate membrane insertion and pore formation. © 2012 Published by NRC Research Press.

Lenka, Sangram K./0000-0002-0121-2430; Elibol Cakmak, Zeynep/0000-0002-6772-5570; Ozkan, Alper/0000-0001-7467-4022 WOS: 000305451400008 PubMed: 22383222 Biodiesel production from microalgae is a promising approach for energy production; however, high cost of its process limits the use of microalgal biodiesel. Increasing the levels of triacylglycerol (TAG) levels, which is used as a biodiesel feedstock, in microalgae has been achieved mainly by nitrogen starvation. In this study, we compared effects of sulfur (S) and nitrogen (N) starvation on TAG accumulation and related parameters in wild-type Chlamydomonas reinhardtii CC-124 mt(-) and CC-125 mt(+) strains. Cell division was interrupted, protein and chlorophyll levels rapidly declined while cell volume, total neutral lipid, carotenoid, and carbohydrate content increased in response to nutrient starvation. Cytosolic lipid droplets in microalgae under nutrient starvation were monitored by three-dimensional confocal laser imaging of live cells. Infrared spectroscopy results showed that relative TAG, oligosaccharide and polysaccharide levels increased rapidly in response to nutrient starvation, especially S starvation. Both strains exhibited similar levels of regulation responses under mineral deficiency, however, the degree of their responses were significantly different, which emphasizes the importance of mating type on the physiological response of algae. Neutral lipid, TAG, and carbohydrate levels reached their peak values following 4 days of N or S starvation. Therefore, 4 days of N or S starvation provides an excellent way of increasing TAG content. Although increase in these parameters was followed by a subsequent decline in N-starved strains after 4 days, this decline was not observed in S-starved ones, which shows that S starvation is a better way of increasing TAG production of C. reinhardtii than N starvation. Biotechnol. Bioeng. 2012; 109:19471957. (c) 2012 Wiley Periodicals, Inc. Turkish Ministry of Agriculture and Rural Affairs General Directorate of Agricultural ResearchGida Tarim Ve Hayvancilik Bakanligi [TAGEM-10/AR-GE/26]; State Planning Organization of the Republic of TurkeyTurkiye Cumhuriyeti Kalkinma Bakanligi Contract grant sponsor: Turkish Ministry of Agriculture and Rural Affairs General Directorate of Agricultural Research; Contract grant number: TAGEM-10/AR-GE/26; Contract grant sponsor: State Planning Organization of the Republic of Turkey

Catestatin (CST), a chromogranin A (CHGA)-derived peptide, is a potent inhibitor of catecholamine release from adrenal chromaffin cells and postganglionic sympathetic axons. We resequenced the CST region of CHGA in an Indian population (n = 1010) and detected two amino acid substitution variants: G364S and G367V. Synthesized CST variant peptides (viz. CST-Ser-364 and CST-Val-367) were significantly less potent than the wild type peptide (CST-WT) to inhibit nicotine-stimulated catecholamine secretion from PC12 cells. Consistently, the rank-order of blockade of nicotinic acetylcholine receptor (nAChR)-stimulated inward current and intracellular Ca2+ rise by these peptides in PC12 cells was: CST-WT &gt, CST-Ser-364 &gt, CST-Val-367. Structural analysis by CD spectroscopy coupled with molecular dynamics simulations revealed the following order of ?-helical content: CST-WT &gt, CST-Val-367, docking of CST peptides onto a major human nAChR subtype and molecular dynamics simulations also predicted the above rank order for their binding affinity with nAChR and the extent of occlusion of the receptor pore, providing a mechanistic basis for differential potencies. The G364S polymorphism was in strong linkage disequilibrium with several common CHGA genetic variations. Interestingly, the Ser-364 allele (detected in ?15% subjects) was strongly associated with profound reduction (up to ?2.1-fold) in plasma norepinephrine/epinephrine levels consistent with the diminished nAChR desensitization-blocking effect of CST-Ser-364 as compared with CST-WT. Additionally, the Ser-364 allele showed strong associations with elevated levels of plasma triglyceride and glucose levels. In conclusion, a common CHGA variant in an Indian population influences several biochemical parameters relevant to cardiovascular/metabolic disorders. � 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Bioprocesses conducted under conditions with restricted O2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative anaerobe Escherichia coli has elaborate sensing and signal transduction mechanisms for redox control in response to the availability of O2 and other electron acceptors. The ArcBA two-component system consists of ArcB, a membrane-associated sensor kinase, and ArcA, the cognate response regulator. The tripartite hybrid kinase ArcB possesses a transmembrane, a PAS, a primary transmitter (H1), a receiver (D1), and a phosphotransfer (H2) domain. Metabolic fluxes were compared under anoxic conditions in a wild-type E. coli strain, its ΔarcB derivative, and two partial arcB deletion mutants in which ArcB lacked either the H1 domain or the PAS-H1-D1 domains. These analyses revealed that elimination of different segments in ArcB determines a distinctive distribution of D-glucose catabolic fluxes, different from that observed in the ΔarcB background. Metabolite profiles, enzyme activity levels, and gene expression patterns were also investigated in these strains. Relevant alterations were observed at the P-enol-pyruvate/pyruvate and acetyl coenzyme A metabolic nodes, and the formation of reduced fermentation metabolites, such as succinate, D-lactate, and ethanol, was favored in the mutant strains to different extents compared to the wild-type strain. These phenotypic traits were associated with altered levels of the enzymatic activities operating at these nodes, as well as with elevated NADH/NAD+ ratios. Thus, targeted modification of global regulators to obtain different metabolic flux distributions under anoxic conditions is emerging as an attractive tool for metabolic engineering purposes. © 2012, American Society for Microbiology. Fil:Ruiz, J.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:de Almeida, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Méndez, B.S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

The dar gene from Lactococcus lactis subsp. cremoris MG1363, encoding the enzyme diacetyl reductase (DAR) was cloned into the expression vector pMG36e under the control of the P32 promoter, and Lc. lactis MG1363 was transformed with this recombinant plasmid by electroporation. Metabolic end products (e. g., pyruvate, lactate, formate, acetoin) of this strain grown in glucose medium were analyzed by HPLC. The overexpression of the plasmid-encoded dar gene in Lc. lactis MG1363 led to the shift from homolactic to mixed acid fermentation and the production of a high level of acetoin, confirming the overexpression of the diacetyl reductase. In addition, the strains overexpressing dar gene and wild type were used as starter cultures in cheese. Different levels of metabolic end products were detected in cheese during the two weeks of ripening compared to the wild type strain.

Adult neurogenesis is maintained by self-renewable neural stem cells (NSCs). Their activity is regulated by multiple signaling pathways and key transcription factors. However, it has been unclear whether these factors interplay with each other at the molecular level. Here we show that SRY-box-containing gene 2 (Sox2) and nuclear receptor tailless (TLX) form a molecular network in adult NSCs. We observed that both Sox2 and TLX proteins bind to the upstream region of Tlx gene. Sox2 positively regulates Tlx expression, whereas the binding of TLX to its own promoter suppresses its transcriptional activity in luciferase reporter assays. Such TLX-mediated suppression can be antagonized by overexpressing wild-type Sox2 but not a mutant lacking the transcriptional activation domain. Furthermore, through regions involved in DNA-binding activity, Sox2 and TLX physically interact to form a complex on DNAs that contain a consensus binding site for TLX. Finally, depletion of Sox2 revealed the potential negative feedback loop of TLX expression that is antagonized by Sox2 in adult NSCs. These data suggest that Sox2 plays an important role in Tlx transcription in cultured adult NSCs., Journal of Biological Chemistry, 287(8), pp.5969-5978; 2012

The plant phenylpropanoid pathway produces an array of metabolites that impact human health and the utility of feed and fiber crops. We previously characterized several Arabidopsis thaliana mutants with dominant mutations in REDUCED EPIDERMAL FLUORESCENCE 4 (REF4) that cause dwarfing and decreased accumulation of phenylpropanoids. In contrast, ref4 null plants are of normal stature and have no apparent defect in phenylpropanoid biosynthesis. Here we show that disruption of both REF4 and its paralog, REF4-RELATED 1 (RFR1), results in enhanced expression of multiple phenylpropanoid biosynthetic genes, as well as increased accumulation of numerous downstream products. We also show that the dominant ref4-3 mutant protein interferes with the ability of the PAP1/MYB75 transcription factor to induce the expression of PAL1 and drive anthocyanin accumulation. Consistent with our experimental results, both REF4 and RFR1 have been shown to physically associate with the conserved transcriptional coregulatory complex, Mediator, which transduces information from cis-acting DNA elements to RNA polymerase II at the core promoter. Taken together, our data provide critical genetic support for a functional role of REF4 and RFR1 in the Mediator complex, and for Mediator in the maintenance of phenylpropanoid homeostasis. Finally, we show that wild-type RFR1 substantially mitigates the phenotype of the dominant ref4-3 mutant, suggesting that REF4 and RFR1 may compete with one another for common binding partners or for occupancy in Mediator. Determining the functions of diverse Mediator subunits is essential to understand eukaryotic gene regulation, and to facilitate rational manipulation of plant metabolic pathways to better suit human needs.

Pseudomonas aeruginosa strains PA7 and Pa5196 glycosylate their type IVa pilins with α1,5-linked D-arabinofuranose (D-Araf), a rare sugar configuration identical to that found in cell wall polymers of the Corynebacterineae. Despite this chemical identity, the pathway for biosynthesis of α1,5-D-Araf in Gram-negative bacteria is unknown. Bioinformatics analyses pointed to a cluster of seven P. aeruginosa genes, including homologues of the Mycobacterium tuberculosis genes Rv3806c, Rv3790, and Rv3791, required for synthesis of a polyprenyl-linked D-ribose precursor and its epimerization to D-Araf. Pa5196 mutants lacking the orthologues of those genes had non-arabinosylated pilins, poor twitching motility, and significantly fewer surface pili than the wild type even in a retraction-deficient (pilT) background. The Pa5196 pilus system assembled heterologous non-glycosylated pilins efficiently, demonstrating that it does not require post-translationally modified subunits. Together the data suggest that pilins of group IV strains need to be glycosylated for productive subunit-subunit interactions. A recombinant P. aeruginosa PAO1 strain co-expressing the genes for D-Araf biosynthesis, the pilin modification enzyme TfpW, and the acceptor PilA IV produced arabinosylated pili, confirming that the Pa5196 genes identified are both necessary and sufficient. A P. aeruginosa epimerase knock-out could be complemented with the corresponding Mycobacterium smegmatis gene, demonstrating conservation between the systems of the Corynebacterineae and Pseudomonas. This work describes a novel Gram-negative pathway for biosynthesis of D-Araf, a key therapeutic target in Corynebacterineae. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Mass spectrometry-based proteomics techniques have been very successful for the identification and study of protein-protein interactions. Typically, immunopurification of protein complexes is conducted, followed by protein separation by gel electrophoresis and in-gel protein digestion, and finally, mass spectrometry is performed to identify the interacting partners. However, the manual processing of the samples is time-consuming and error-prone. Here, we developed a polymer-based microfluidic proteomic reactor aimed at the parallel analysis of minute amounts of protein samples obtained from immunoprecipitation. The design of the proteomic reactor allows for the simultaneous processing of multiple samples on the same devices. Each proteomic reactor on the device consists of SCX beads packed and restricted into a 1 cm microchannel by two integrated pillar frits. The device is fabricated using a combination of low-cost hard cyclic olefin copolymer thermoplastic and elastomeric thermoplastic materials (styrene/(ethylene/butylenes)/styrene) using rapid hot-embossing replication techniques with a polymer-based stamp. Three immunopurified protein samples are simultaneously captured, reduced, alkylated, and digested on the device within 2-3 h instead of the days required for the conventional protein-protein interaction studies. The limit of detection of the microfluidic proteomic reactor was shown to be lower than 2 ng of protein. Furthermore, the application of the microfluidic proteomic reactor was demonstrated for the simultaneous processing of the interactome of the histone variant Htz1 in wild-type yeast and in a swr1Δ yeast strain compared to an untagged control using a novel three-channel microfluidic proteomic reactor. © 2011 American Chemical Society.

Reporter gene transactivation by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. To investigate the role of thioredoxin in controlling p53 activity, the level of reporter gene transactivation by p53 was determined in yeast lacking the TRX1 and TRX2 genes encoding cytosolic thioredoxin. Surprisingly, p53 activity was unimpaired in yeast lacking thioredoxin. Subsequent analyses showed that thioredoxin deletion suppressed the inhibitory effect of thioredoxin reductase deletion, suggesting that accumulation of oxidized thioredoxin in mutant yeast was necessary for p53 inhibition. Purified human thioredoxin and p53 interacted in vitro (Kd = 0.9 microM thioredoxin). To test the idea that dithio-disulfide exchange reactions between p53 and thioredoxin were responsible for p53 inhibition in mutant yeast, each p53 cysteine was changed to serine, and the effect of the substitution on p53 activity in TRR1 and Deltatrr1 yeast was determined. Substitutions at Zn-coordinating cysteines C176, C238, or C242 resulted in p53 inactivation. Unexpectedly, substitution at cysteine C275 also inactivated p53, which was the first evidence for a non-zinc-coordinating cysteine being essential for p53 function. Cysteine substitutions at six positions (C124, C135, C141, C182, C229, and C277) neither inactivated p53 nor relieved the requirement for thioredoxin reductase. Furthermore, no tested combination of these six cysteine substitutions relieved thioredoxin reductase dependence. The results suggested that p53 dependence on thioredoxin reductase either was indirect, perhaps mediated by an upstream activator of p53, or was due to oxidation of one or more of the four essential cysteines.

The current strategy to improve the quality of life of Human Immunodeficiency Virus (HIV) infected individuals through suppressing viral replication and maintaining the virus at low to undetectable levels is based on highly active antiretroviral therapy (HAART). Protease inhibitors are essential components of most HAART protocols and are often used as the first line of treatment. However, a considerable percentage of new HIV-1 infections are caused by viruses carrying antiretroviral drug-resistant mutations. In this paper molecular dynamics, docking simulations, and free energy analysis of mutated HIV protease complexes were used to estimate the influence of different drug resistance-associated mutations in lopinavir, amprenavir, saquinavir, and atazanavir protease recognition. In agreement with virological and clinical data, the structural analysis showed that the single mutations V82A, I84V, and M46I are associated with higher energetic values for all analyzed complexes with respect to wild-type, indicating their decreased stability. Interestingly, in atazanavir complexes, in the presence of the L76V substitution, the drug revealed a more productive binding affinity, in agreement with hypersusceptibility data.

Mutagenesis of specific amino acid residues of the glucansucrase (GTF180) enzyme from Lactobacillus reuteri strain 180 yielded 12 mutant enzymes that produced modified exopolysaccharides (mEPSs) from sucrose. Ethanol-precipitated and purified mEPSs were subjected to linkage analysis, Smith degradation analysis, and 1D/2D 1H NMR spectroscopy. Comparison of the results with structural data of the previously described wild type EPS180 and triple mutant mEPS-PNNS revealed a broad variation of structural elements between mEPS molecules. The amount of (α1→3) linkages varied from 14-43%, the amount of (α1→4) linkages (not present in the wild type) from 0-12%, and the amount of (α1→6) linkages from 51-86%. The average molecular weight (Mw) ranged from 9.4 to 32.3 MDa and the degree of branching varied from 8-20%. Using a previously established 1H NMR structural-reporter-group concept, composite models, that include all identified structural features, were formulated for all mEPS molecules. Variations in the mEPS structures strongly affected the physical properties of the mEPSs. © 2009 American Chemical Society.

Mutagenesis of specific amino acid residues of the glucansucrase (GTF180) enzyme from Lactobacillus reuteri strain 180 yielded 12 mutant enzymes that produced modified exopolysaccharides (mEPSs) from sucrose. Ethanol-precipitated and purified mEPSs were subjected to linkage analysis, Smith degradation analysis, and 1D/2D 1H NMR spectroscopy. Comparison of the results with structural data of the previously described wild type EPS180 and triple mutant mEPS-PNNS revealed a broad variation of structural elements between mEPS molecules. The amount of (α1→3) linkages varied from 14-43%, the amount of (α1→4) linkages (not present in the wild type) from 0-12%, and the amount of (α1→6) linkages from 51-86%. The average molecular weight (Mw) ranged from 9.4 to 32.3 MDa and the degree of branching varied from 8-20%. Using a previously established 1H NMR structural-reporter-group concept, composite models, that include all identified structural features, were formulated for all mEPS molecules. Variations in the mEPS structures strongly affected the physical properties of the mEPSs. © 2009 American Chemical Society.